ABSTRACT Chronotype, or morningness-eveningness preference, is a behavioral manifestation of an individual?s underlying timing of the circadian rhythm relative to environmental cues and the molecular basis of its links to metabolism remains poorly understood. N-glycosylation is a post-translational modification that is important for the folding, stability and secretion of proteins, and our recent findings implicate causal roles for N-glycosylation changes in chronotype. Our recent collaborative GWAS in 697,828 participants from the UK Biobank and 23andMe identified 351 loci for self-reported chronotype, including at ALG10 and ALG10B, paralogous genes that encode enzymes required for addition of the final glucose residue to assembling N-glycan precursors in the endoplasmic reticulum lumen. Pathway analyses confirmed enrichment of other N-glycosylation pathway enzymes in chronotype GWAS, reinforcing the importance of this pathway. Furthermore, ALG10B genetic variants were found in our published GWAS of self-reported sleep duration, and in GWAS of objective measures of sleep and activity timing in 85,760 UK Biobank participants with 7-day accelerometry. Functional follow-up by modeling ALG10 knockdown in Drosophila brain identified sleep disturbances and epilepsy. Here we propose to use and adapt glycoscience tools developed under the NIH Common Fund Glycoscience Program to determine the molecular consequences on the glycoproteome of perturbation of ALG10B/ALG10 enzymes and to better understand the connections between circadian rhythms and N- glycosylation in both human cellular models and the Drosophila brain. This work will directly expand understanding of human relevant molecular mechanisms that drive timing of the internal circadian rhythm (Aim 2 of the parent grant) and its links with metabolism.